The development of a technology of producing a chimeric animal by fusing micronuclei comprising human chromosome fragments with cells having pluripotent differentiation to obtain hybrid cells allows to prepare a non-human animal maintaining large exogenous genes to be produced (Non-Patent Document 1 and Patent Document 1).
Subsequently, a method for constructing a desired human artificial chromosome (hereinafter abbreviated to HAC) vector to produce the non-human animal has been proposed, by which the HAC including a wide range of human antibody gene loci has been established.
First, as a method for modifying a chromosome fragment to be introduced into a non-human animal, a technology of preparing a deleted chromosome with a high efficiency by inserting a telomere sequence into a desired sequence on a human chromosome kept in a chicken DT40 cell by gene targeting has been developed (Patent Document 2)
Further, in a process of maintaining a mouse cell keeping a human chromosome, it has been found that a fragment SC20 comprising an antibody heavy chain gene locus derived from the human chromosome 14 may be obtained, and the fragment is stably maintained in an embryonic stem (ES) cell and an individual of the mouse and has high transmission efficiency to progeny (Patent Document 2).
Therefore, a λHAC comprising a human antibody heavy chain and a human antibody λ chain was constructed by translocating a fragment comprising the antibody type light chain gene locus on the human chromosome 22 through the Cre/loxP site specific recombination system using such SC20 as a basic skeleton of a vector (Non-Patent Document 2 and Patent Document 3).
The λHAC has the stability and transmission efficiency to progeny almost equivalent to the SC20 and a chimeric mouse which stably maintains a λHAC is produced by introducing the λHAC into the mouse ES cells (Non-Patent Document 2 and Patent Document 3). It is now possible to construct a HAC vector including a human chromosome region having a specific megabase (Mb) size by the method.
Further, for the purpose of removing chromosome regions which adversely affect the generation of a chromosome-introduced animal, chromosome fragments ΔHAC and ΔΔHAC with an optimal size including the antibody λ type light chain (λ chain) gene region were prepared (Patent Document 4), based on the structural information of the human chromosome 22 (Non-Patent Document 3).
It was confirmed that the ΔHAC and ΔΔHAC include regions having 2.5 Mb and 1.5 Mb sizes, respectively, which are shorter than the periphery of the antibody λ type light chain gene region on the MAC, 10 Mb, to impart a transmission efficiency to progeny higher than that of the λHAC (Patent Document 4).
Meanwhile, human polyclonal antibodies currently used for treatment and prevention of various diseases are prepared from a serum pool obtained from a plurality of human donors. For this reason, the performance of the human polyclonal antibodies depends largely on human donor sera as a supplying source, and thus a process of selecting a donor having a desired antigen reactivity or titer is required in preparing.
In addition, due to factors such as the kind of antigen, the number of exposure to an immunogen, and the amount of donor serum which may be collected, in preparing a polyclonal antibody, the development of the use thereof is limited. Therefore, a transgenic animal having a human antibody gene locus as a means for producing human polyclonal antibodies has been prepared.
Due to its body size, an ungulate animal is useful as a source of supplying a large quantity of human polyclonal antibodies. So far, in order to produce human polyclonal antibodies, a bovine into which the above-described HAC, specifically ΔHAC and ΔΔHAC are introduced to produce human polyclonal antibodies, is known (Non-Patent Document 4 and Patent Document 5).
In the neonatal sera of the bovine into which these HACs were introduced, 13 to 258 ng/mL of the human immunoglobulin (Ig) G was produced (Non-Patent Document 4).
Subsequently, in order to eliminate bovine antibodies produced in a bovine living body, gene targeting was carried out on IGHM and IGHML1 encoding the functional IgM genes among bovine endogenous antibody heavy chain genes and as a result, a bovine in which antibody heavy chains were knockout was known (Non-Patent Documents 5 and 6 and Patent Documents 6 and 7).
When ΔΔHAC was introduced into the obtained antibody heavy chain double-knockout bovine (IgHM−/−/IgHML1−/− bovine), it was found 7.1 μg/mL of a human IgG was produced in a serum of a 14-day-old calf (Patent Document 7).
In addition, a bovine into which κHAC which is a HAC vector having a human antibody heavy chain gene locus and a human antibody κ type light chain (κ chain) gene locus was introduced was produced (Non-Patent Document 6 and Patent Document 8). κHAC is a vector constructed by translocating a fragment comprising the antibody κ chain gene locus on the human chromosome 2 onto the SC20. In FIG. 1, a schematic view of κHAC is shown.
Among IgHM−/−/IgHML−/− bovines into which κHAC was introduced, clone 468 which was the highest antibody producing individual constantly produced a human IgG at 1 g/L or more in the serum from the 84 days after birth, and exhibited a titer exceeding 2 g/L at the 210 days after birth.
However, a technology of stably producing an individual which exhibits high titer as described above has not been known, and thus there is a need for animals which produce human antibodies with higher efficiency or a technology which can stably produce these animals.